超高效液相色谱-串联质谱法测定亚硫酸氢钠穿心莲内酯在大鼠肝微粒体中的含量

目的: 建立超高效液相色谱-串联质谱法测定大鼠肝微粒体中亚硫酸氢钠穿心莲内酯。 方法: 以肝微粒体孵育液为基质,通过添加标准溶液的方法配制含亚硫酸氢钠穿心莲内酯和内标物大豆苷元的样品,选用甲醇为沉淀剂,经离心除去肝微粒体孵育液中的蛋白质,上清液用于目标物的检测。采用Thermo Hypersil Gold C₁₈分析柱(50 mm×2.1 mm,1.9 μm),预柱:Phenomenex Security Guard C₁₈(4 mm×3.0 mm),以甲醇-水溶液为流动相,梯度洗脱,流速:0.2 mL·min^-1,柱温:35℃,整个分析时间为6 min。采用电喷雾负离子(ESI^-)模式电离,选择反应监测(SRM)模式检测,以大豆苷元作为内标物质进行定量。用于监测的离子分别为亚硫酸氢钠穿心莲内酯m/z 413.2 → 287.2/331.2和大豆苷元m/z 253.1→132.2,用基质匹配标准溶液法进行定量。 结果: 肝微粒体孵育液中亚硫酸氢钠穿心莲内酯的质量浓度在0.05 ~ 50 μmol·L^-1范围内线性良好(r = 0.9977,权重系数W: 1/x²);孵育液中亚硫酸氢钠穿心莲内酯的检出限(信噪比为3)为0.02 μmol·L^-1;其平均回收率在85%~115%之间;日内及日间的相对标准偏差(RSD)均小于15%,满足生物样品检测的要求。 结论: 将该方法用于肝微粒体孵育液中亚硫酸氢钠穿心莲内酯的检测。该方法选择性强、灵敏度高、操作简便快速、重现性好,适用于亚硫酸氢钠穿心莲内酯体外代谢动力学等方面的研究。

Objective: To establish an ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC - MS/MS) method for the determination of andrographolide sodium bisulphite (ASB) in rat liver microsomes. Methods: The inactivated liver microsomes incubation medium was used as a substrate,ASB samples and the internal standard (daidzein) were prepared by adding standard solution.Methanol was added in the sample for the deproteinization.Then the sample was vortex-mixed and centrifuged.The clear supernatant was used for the analysis of UHPLC-MS/MS.A Thermo Hypersil Gold C₁₈ column (50 mm×2.1 mm,1.9 μm) was employed with a guard column of Phenomenex Security Guard C₁₈ column (4 mm×3.0 mm),and the column temperature was set at 35℃.The gradient elution was performed at a flow rate of 0.2 mL·min^-1 with methanol and water as mobile phases,and a rapid separation was completed in 6 min.The electrospray was operated in the negative ionization mode,the ASB and daidzein were identified by selected reaction monitoring (SRM) mode,and the monitoring ions of them were m/z 413.2→287.2/331.2 and m/z 253.1→132.2,respectively,which were used to qualify and quantify the samples by the method of matrix-matched standard solution. Results: The calibration curve showed good linearity within the concentrations of 0.05 to 50 μmol·L^-1 (r = 0.9977,Weighing: 1/x²); the limit of detection (S/N=3) was 0.02 μmol·L^-1.The mean recoveries were from 85% to 115% at the spiked levels of 0.1,2 and 40 μmol·L^-1; the relative standard deviations (RSDs) of intra- and inter-day of variation were both less than 15%,which can meet the determination requirements of biological samples. Conclusions: The method was initially used for the determination of andrographolide sodium bisulphite in liver microsomes.The method is selective,sensitive,convenient,rapid and reproducible in the determination of ASB,which can be used for the kinetics research of ASB in vitro.